1. Field of the Invention
The invention relates generally to the preparation of chromosome specific DNA probes that are useful in selectively detecting individual chromosomes or chromosome segments. The method may be used for detection of chromosomes or chromosome segments in metaphase cell spreads and in interphase nuclei by in-situ hybridization.
2. Description of Related Art
Chromosome identification procedures have long been considered an integral aspect of biomedical diagnostic practice because of the many disease syndromes having a genetic basis which are caused or diagnosed by chromosomal alterations. Being able to observe these chromosomal alterations in cells of the body, therefore, not only helps in the diagnosis of the disease but has the potential of being an effective monitor of therapeutic procedures designed to eliminate the cells with the chromosomal anomalies from the body. It is also important to be able to rapidly monitor the chromosomes of the fetuses of pregnancies at risk (such as in older women, or where a parent may have been exposed to environmental mutagen, or where previous siblings of the fetus have an abnormality with known chromosomal basis). Specific chromosomal abnormalities, translocations (exchanges between the arms of two different chromosomes), inversions (internal segment of a chromosome swings around and faces the opposite direction), deletions (piece of chromosome becomes lost), monosomies (only one instead of two chromosomes of a type), and trisomies (three instead of two chromosomes of a type) have been associated with specific sub-types of cancer (1) as well as in a wide variety of inherited or congenital abnormalities (2).
Chromosomally based disease syndromes are often seen in hematological malignancies such as leukemia and lymphoma. The classic example is the 9:22 translocation associated with chronic myelogenous leukemia (CML). Here, there is a reciprocal translocation between the end of the long arm of chromosome 9 and the middle of the long arm of chromosome 22 producing what is known as the Philadelphia (Ph) chromosome of the translocation chromosome carrying the chromosome 22 centromere (3-5). The ability to see such events was made possible by a staining method developed in the early '70s which imparted to each of the 24 different human chromosomes a distinctive banding pattern (6,7). Therefore, by carrying out such a stain on cells and photographing them, individual chromosomes can be cut out and lined up (according to their bands) in a procedure called karyotyping. By doing this the translocation, or any abnormal chromosome eventually stands out from the rest and is identified.
While effective and important in original diagnosis, this procedure is impractical for determining the percentage of such cancer cells in patients with minimal residual disease or in relapse because it is arduous and labor-intensive. Furthermore, with respect to monitoring pregnancies, a faster method is preferred. A disadvantage of the procedure is its dependence on high quality metaphase spreads (structures formed when the chromosomes condense and form their distinctive morphology and banding pattern during the division phase of the cell cycle). Good, scorable metaphases are often not available in the tissues which one needs to monitor (e.g., bone marrow which is the source of many leukemic cells, or amniocentesis tissues for fetus evaluation). The result is that rearranged chromosomes are often not identifiable by this method leaving the entities of unknown origin to be referred to as "marker chromosomes".
Two groups (8,9) have described a method to stain a specific chromosome of choice. The problem associated with such a procedure was to obtain DNA from only the chromosome of interest, label it in some way so that it would be identified later (this labeled DNA is therefore chromosome-specific "probe"), and then hybridize probe to the chromosomes in a cell on a microscope slide. By visualizing the label, the chromosome was visualized since the DNA, if properly handled, hybridized only to the chromosome from which it was derived. The procedure has since come to be known as chromosome painting. Using a human chromosome 21 probe, trisomies and translocations associated with the chromosome (9,10) were visualized even in poorly defined metaphases. Specific chromosomes of interest used to prepare painting probes have been separated from other chromosomes by flow-sorting synchronized populations of dividing cells through a technically rigorous procedure requiring highly specialized and expensive equipment (a fluorescence-activated cell sorter). This leads to only a small quantity of material, so that the DNAs from the individual chromosomes then need to be extracted and cloned into cosmid libraries. To make probe, the cosmid library must be expanded, DNA extracted again and nick-translated in the presence of biotinylated nucleotide prior to hybridization to human metaphase spreads and detection of the specific chromosomes (11). An essential requirement for specific chromosome painting is the prehybridization of the probe with total human DNA in order to prevent human repeat sequences (which are not chromosome specific) from participating in the in-situ hybridization reaction.
Unfortunately, flow sorting of individual chromosomes, making and expanding and maintaining cosmid libraries is problematical because of possible contamination with other chromosomes or the presence of non-chromosomal specific sequences in such material that cannot be prevented from participating in the in-situ hybridization. These concerns have been recently highlighted (12) in studies where such probe made for human chromosome 22 hybridized to additional chromosomal regions on human metaphases. Other serious limitations of the approach include: 1) cross-hybridization of the chromosome 2 probe to the centromere of chromosome 19; 2) failure of libraries made from chromosome 5 to paint chromosome 5; 3) cross-hybridization of probes made from flow-sorted chromosomes 13, 14, 15, 21, and 22 to the centromeric regions of each other; and 4) cross-hybridization of the chromosome 18 probe to the centromeric regions of chromosomes 12 and 19 (as in poster presentation at the 41st Annual Meeting of the American society of Human Genetics--13). The method is also expensive and limited to the availability of the flow sorted libraries. The procedure is relatively inflexible. For instance, there are situations in which one might be interested in painting only a portion of a specific chromosome (e.g., the p-arm of chromosome 16). This has not yet been achieved using flow-sorted libraries, yet this would be an excellent probe for the identification of inversions associated with cancer and other disease syndromes. An example of this is acute nonlymphocytic leukemia where there is a pericentric inversion involving both the short and long arms of chromosome 16 (14). Probe for only the short arm of the chromosome would identify the inversion chromosome as one which had staining on portions of both arms whereas a normal chromosome 16 would have only the short arm painted. Finally, probe made from flow sorted chromosome libraries does not allow the identification of the regions of the respective chromosomes brought together by the rearrangement because the longitudinal differentiation (banding) of the specific chromosome is lost and probes to paint specific portions of chromosomes are not available by this method. Therefore, the approach is not effective in identifying either break point sites in rearrangements or deletions (or other events in which only a single chromosome is affected).
Another way to isolate human chromosome-specific DNA is amplification by polymerase chain reaction (PCR) from interspecific hybrid cells containing only the human chromosome or chromosomal region of interest. Human-rodent hybrid cells monochromosomal for virtually every human chromosome and for portions of a human chromosome are now available. Since it is known (15) that the human genome has hundreds of thousands species-specific repeat sequences scattered throughout, several groups (16-19) have prepared consensus primers to bind to these sequences. By using these primers for PCR amplification, some success in pulling out human chromosome specific DNA sequences from hybrid cells and various types of recombinant DNA libraries have been achieved. However, a recently reported attempt to make painting probe from such material (20) resulted in speckled chromosomes with high background with no possibility of observing any longitudinal differentiation of the painted genetic element. Although selective in amplifying human DNA from hybrid cells, sensitivity in chromosome painting was low. Thus, the reported methods are impractical for developing probe useful for the identification of deletions or translocation breakpoints in abnormal cells.
An attempt to paint specific human chromosomes with the total DNA from a hybrid cell containing the human chromosome of interest has met with partial success (21). Unfortunately, for most of the chromosomes attempted, no clear specific painting was obtained. In the few cases where success in painting a specific chromosome was achieved, the entire chromosome was painted and there was no possibility of observing longitudinal differentiation.
Identification of cells having chromosomal rearrangements at known breakpoints on the affected chromosomes has been accomplished by using, as probe, sets of cosmids that flank the breakpoint. Use of such cosmid probes flanking the breakpoint region of the p-arm of chromosome 16 has enabled visualization of the inversion associated with acute nonlymphocytic leukemia (22). However, the intensity of the signal was relatively weak. One would expect a much stronger signal if the entire arm of the chromosome were painted. Using a similar technique, the CML Ph chromosome has been identified using a pair of cosmids, one from the brc gene proximal to the breakpoint on chromosome 22 and the other from the abl gene distal to the breakpoint on chromosome 9 (23). The intensity of the signal appeared weak, raising doubt that it could be reliably used to identify the breakpoint in the majority of cells without computer enhancement. These latter two approaches, while somewhat effective for chromosomal alterations where genomic regions flanking the breakpoints have been cloned, are useful only where such sites have been precisely identified or isolated. In the vast majority of cases, the sites are unknown and there is no effective method of identification.
Finally, DNA probes have been developed that specifically detect the centromeres (the dot-like structure at the junction of the two chromosome arms) for particular chromosomes (24). These probes are supposed to be effective in determining the number of chromosomes (for chromosomes from which such probes have been developed) in cells (24). However they provide no information on rearrangements involving those chromosome. Additionally, they often lack specificity in that probe developed for the identification of one centromere will often cross-hybridize to centromeres of other chromosomes.
Some progress has been made in developing techniques to selectively identify individual human chromosomes. However, current methods have several shortcomings, including: (1) lack of detection of abnormal chromosomes in less than ideal metaphase spreads; (2) impracticality in determining the frequency of abnormal cells in a complex tissue; (3) inefficient detection of identify of marker chromosomes; (4) lack of specificity in identifying sub-chromosomal regions; (5) great time and expense involved in either karyotyping or preparing probe; (6) lack of flexibility; and (7) failure to paint chromosomes adequately while still observing landmarks for longitudinal differentiation.